1. Field of the Invention
The present invention relates to a silver nano-structure for surface enhanced Raman scattering (SERS) substrate and silver nano-structure thereby.
2. Description of the Related Art
High sensitivity detection of biological sample or other samples to the level of single molecule detection can be widely utilized in a variety of areas including diagnostics, pathology, toxicology, environmental sampling, or chemical analysis. For this purpose, nano-particles and chemicals labeled with specific substances have been widely used in researches on metabolism, distribution and binding of small amount of synthetic materials and biomolecules. For example, radioisotopes, organic fluorescent dyes or inorganic quantum dots have been widely used.
The method using radioisotopes generally uses 3H, 14C, 32P, 35S, or 125I which are radioactive isotopes of 1H, 12C, 31P, 32S, or 127I. The radioisotopes have almost identical chemical properties as non-radioactive isotopes and thus are arbitrarily substitutable, and their relatively large emission energy enables even a small amount detection. For the above advantages, the radioisotopes have been used for a long period of time. However, using radioisotopes also has shortcomings. That is, it is not easy to conduct the process because of harmful radiation, and it is not convenient to store or conduct experiment with certain isotopes despite large emission energies, particularly those with short half-lives.
One of the representative replacements for the radioisotopes is the organic fluorescent dyes. The organic fluorescent dyes emit light with unique wavelength, as these are excited by the light at certain wavelengths. As the detection methods pursue minimization, radioactive materials showed limited sensitivity, taking long time until detection result is obtained. Compared to this, fluorescent dyes can emit several thousands of photons per molecule when placed under proper conditions, and in theory, it can detect even a single molecule level.
However, using fluorescent dyes also has shortcomings. That is, unlike radioisotopes, it is not possible to directly substitute the atoms of the active ligand, and it is required to connect the fluorescent dyes by distorting certain portions that have relatively less influence on activity based on structure-activity relationship. The fluorescent dyes suffer photobleaching as time passes, and very narrow light wavelength of activation and very wide wavelength of the light emission cause interferences between different fluorescent dyes. Furthermore, the number of available fluorescent dyes is limited.
The quantum dots, which are semiconductor nano materials, are composed of CdSe, CdS, ZnS, ZnSe and emit different colored lights depending on sizes and types. Compared to organic fluorescent dyes, the quantum dots have broader activity wavelengths, while these represent narrow light emission wavelength. Accordingly, there are more quantum dots than organic fluorescent dyes that emit different colors. For the above reasons, quantum dots are more generally used recently, as a method for overcoming drawbacks of the organic fluorescent dyes. However, the quantum dots also have shortcomings such as high toxicity and other constraints that prevent mass production. While there are a variety of quantum dots theoretically, in actual use, the number of quantum dots that can be used is quite limited.
The Raman spectroscopy and/or labeled substances for surface plasmon resonance have thus been suggested to resolve the problems mentioned above.
The surface enhanced Raman scattering (SERS) is the spectroscopic method that utilizes abrupt surge of Raman scattering intensity by more than 106˜108 times higher, where the molecules are adsorbed on roughened surfaces of metal nano-structures such as gold or silver. When the light passes a concrete medium, a certain amount of different types of lights are generated other than the original wavelength of the light, which is the Raman scattering. Some of the scattered lights have varied frequencies from the originally excited light as the vibrating state of the molecules are excited to higher energy level, and the Raman scattering spectrum wavelengths represent chemical composition and structural properties of the light-absorbing molecules in the sample. Accordingly, Raman spectroscopy, in combination with the fast-advancing nano technology, is anticipated to be the future technology that can directly measure single molecules with high sensitivity, and also as the essential tool for medical sensing. The SERS effect is associated with Plasmon resonance phenomenon, in which metal nanoparticles show clear optical resonance in response to incident electromagnetic radiation due to collective coupling among the conductive electrons within the metal. Basically, the nanoparticles of gold, silver, copper and certain other metals can act as a small antenna to enhance centralization of the electromagnetic radiations. Molecules located adjacent to such particles show considerably higher sensitivity than ordinary Raman scattering.
Therefore, researches have actively conducted to perform early diagnosis of genes or proteins (i.e., biomarkers) associated with a variety of disease, using SERS sensors. The Raman spectroscopy provides several advantages that other analysis methods (including infrared spectroscopy) cannot. Compared to infrared spectroscopy which can obtain strong signal only from the molecule with dipole moment, Raman spectroscopy can obtain strong signals even from non-polar molecules which have variations in induced polarizability, which means all the organic molecules have their own Raman shifts (cm−1). In addition, Raman spectroscopy is free from the interference by water molecules, and thus is more suitable for the detection of biomolecules such as proteins or genes. However, the relatively low signal strength hinders the practical use of the Raman spectroscopy despite the long period of researches.
Following the discovery of SERS, researches continued. After the report about SERS which is capable of signal detection in a single molecular level in chaotic aggregates of nanoparticles to which fluorescent molecules are adsorbed (Science 1997, 275(5303), 1102; Phys. Rev. Lett. 1997, 78(9), 1667), researchers reported about SERS potentiation using a variety of nano-structures (nanoparticles, nanoshells, nanowires). To utilize the high sensitivity SERS phenomenon in the development of biosensors, Mirkin Research Group (Northwestern Univ.) has recently successfully conducted high sensitivity DNA analysis using DNA-gold nanoparticles, with the detection limit of such format reaching 20 fM (Science, 2002, 297, 1536). However, none has shown any progress since the initial research about method for preparing single molecule SERS substrate based on salt induced aggregation of silver (Ag) nanoparticles with Raman-active molecules (e.g., Rhodamine 6G). Report said that only fraction (below 1%) of the colloids with heterogeneous aggregation has single molecular SERS activity (J. Phys. Chem. B 2002, 106(2), 311). Although the random heterogeneous (i.e., roughened) surfaces provide a great amount of interesting and essential data associated with the SERS, such strategy is basically not reproducible, because the enhancement is subject to considerable change even by a small change on surface morphology. Recently, Fang et al. reported about quantitative measurement on distribution of enhancement in SERS. While the most concentrated parts (EF>109) were 64 out of total 1,000,000, such only contributed to 24% of the total SERS intensity (Science, 2008, 321, 388). If any structure that can maximize SERS signal is ensured, very useful, high-sensitivity, and high-reliability biomolecule analysis will be made available. This will be also very useful for imaging technology both in vitro and in vivo.
Most SERS detections or, various analytes used colloidal metal particles coated on substrate and/or supports such as aggregated silver nanoparticles. However, while such arrangement enables SERS detection with sometimes 106 to 108 times greater sensitivity, this cannot detect single molecule of the small analyte such as nucleotide. Despite the advantages of SERS, the SERS phenomenon has incompletely elucidated mechanism, and attempts to develop and commercialize nano-biosensors and to apply the SERS phenomenon are still faced with many challenges that have to be tackled with, such as lack of accurate, structurally-defined nanoparticle synthesis and control, and difficulty in reproducibility and reliability due to variation of enhancement efficiency depending on wavelength of the light for use in spectrum measurement, or direction of polarization. Therefore, to resolve the problems explained above, accurate understanding of optical properties of well-defined nano-structures and accurate control on SERS phenomenon at the same time is necessary.
L. Brus et al. (JACS. 2002) reported that SERS signal was enhanced in the metal particle dimmers, when very strong electromagnetic field (hot spot or interstitial field) was formed between two or more nanoparticles, and according to electromagnetic theory calculation, approximately 1012 SERS enhancement is expected from the hot spot. The enhanced sensitivity of Raman detection varies depending on presence or absence of the hot spot, although it is not clearly regular in the colloid particle aggregation. However, none has suggested about the relationship between the physical structure of the hot spot, distance range from the nanoparticles with enhanced sensitivity, and spatial relationship between the analyte to enhance the sensitivity and nanoparticle aggregations. Furthermore, the nanoparticle aggregation is basically unstable in solution, and gives adverse effect on the reproducibility of the single molecular analyte detection.
By amplifying the optical signal, the electromagnetic signals at the external junctions on two or more nano-structures are amplified, thus enabling detection of unique amplified signals (e.g., Raman, fluorescence, scattering) of the molecules at the gap emitting optical signals. However, to obtain SERS signals using such structures, issues like signal quantification, reproducibility of the result, convenience and simplicity of synthesis, cost or stability of probe still remain as the problems to be tackled with.
From the microscopic prospect, the fact that different physical properties appear depending on sizes and shapes of particles can mean hindrance to infinite utilization in the application fields like catalysts, nano devices, nano sensors or medicines. Accordingly, researches focus on inventing nano-structures that are accurately defined structurally. Currently, many preparation methods are available for preparing nanoparticle colloid with regular size distribution and high dispersive powder in 10-100 nm range.
However, it is still necessary to invent SERS substrate with high-sensitivity SERS activity and reproducibility, from the prospect of principles of spectroscopic structure analysis or detection with SERS on molecules adsorbed to metal surfaces. The principle that explains SERS effect is generally categorized into mainly, electromagnetic theory and charge transfer theory. The electromagnetic theory car, be explained with local field enhancement. That is, upon exposure to light that meets plasmon resonance conditions of gold, or silver nanoparticles, the electromagnetic field of electromagnetic radiation is amplified near the surface of the metal nanoparticle. The adsorbed molecules have amplification of Raman scattering due to enhanced electromagnetic radiation. On the other hand, the charge transfer theory is based on chemical enhancement, according to which the metal-molecule complex in charge transferable state theoretically act as resonant condition which mediates resonant Raman scattering. The intensity of SERS mainly relies on the resonance structure of surface plasmon, which is determined according to metal nano-structures. Accordingly, it is necessary to design nano-structures that can increase SERS cross sectional area and to enhance reproducibility.
The SERS phenomenon is comprehensively understood in its mechanism or aspect of spectroscopic structure analysis on adsorbate. The general SERS spectrum for measurement characteristically represents molecules adsorbed on the metal surface. Accordingly, SERS is done mainly to determine orientation of the molecules adsorbed to the nano-structures or vibrational structure thereof, based on the analysis on size of enhancement or vibrational mode (peak intensities and locations) rather than analysis on peak profiles by metal nanoparticles. Generally, gold nanoparticles provide larger SERS cross section area than silver nanoparticles, and better reproducibility. However, due to less competitive price of the gold nanoparticles, demands increases for silver nano-structures with high sensitivity SERS activity and reproducibility. Accordingly, silver nanoparticle colloids are considered to be a good candidate for SERS substrate development. A silver nano-structure preparation method is thus necessary, which can provide silver nano-structures with high sensitivity SERS activity in both adsorbate spectroscopic structural analysis and principle of detection and reproducibility, and which can provide silver nano-structures with hot spots having large SERS cross section areas based on the same.
The present inventors discovered that, in the conventional silver nanoparticle preparation method using AgNO3 aqueous solution and NaBH4 reductant, by characterizing a variety of unpredictable conditions such as, concentration of AgNO3 and reductant, reaction temperature, stirring velocity, single dropwise addition quantity, dropwise addition rate, or total dropwise addition quantity, it is possible to ensure the ‘hot spot’, the considerably very intense electromagnetic field in which two to four particles are agglomerated in the prepared silver nano-structures in such a form to enhance SERS signals, and also to provide uniform-sized silver nano-structures and provide reproducibility of the silver nano-structures, and therefore, completed the present invention based on such finding.